The spectrum of genetic diseases that can be therapeutically addressed by using lentiviral vectors is restricted by their oncogenic potential, which is inherent to their integrative nature. In this study we propose to develop and utilize novel non-integrating lentiviral vectors to address the above obstacle. Our approach is based on recent findings in our laboratory demonstrating the ability of the HIV-1 Rev/RRE system to function as a secondary packaging system, which mediates packaging of non-HIV-1 mRNAs into HIV-1 particles. We showed that HIV-1 RRE containing EIAV vectors efficiently packaged into HIV-1 particles. The novel chimeric vectors, which lacked the ability to integrate into a host cells' genome, exhibited transgene expression levels significantly higher than other non-integrating vectors, such as HIV-1 vectors packaged with the HIV-1 integrase mutant E152A. Here we propose to: i) characterize the mechanism involved in the Rev/RRE dependent packaging of chimeric EIAV/HIV-1 vectors; ii) characterize the ability of the HIV-1 to process the EIAV att sites and its effects on chimera vector integration; iii) investigate the effects of histone modifications and chromatin structure on transgene expression from EIAV/HIV-1 chimera vectors; iv) to characterize the biodistribution of the chimera EIAV/HIV-1 vectors; and v) to determine the efficacy of the chimera vectors at delivering and maintaining high levels of human factor IX expression in a humanized hemophilia mouse model We believe that the proposed studies will result in the development of efficacious non-integrating lentiviral vectors, which will be better suited for human gene therapy. [unreadable] [unreadable] [unreadable]